Comparison of culture and 2 real-time polymerase chain reaction assays to detect group B Streptococcus during antepartum screening

Group B Streptococcus (GBS) is the most common cause of life-threatening infection in neonates but is preventable if the mother is diagnosed before and treated at delivery. Using 200 vaginal-rectal swabs inoculated to enrichment (LIM) broths, we compared routine culture and 2 real-time polymerase chain reaction (PCR) assays for detection of GBS: the LightCycler (LC) Strep B analyte-specific reagents (ASRs) (Roche Diagnostics, Indianapolis, IN) and the BD GeneOhm StrepB (BD-StrepB) test (BD GeneOhm Sciences, San Diego, CA). Culture detected 26.5% GBS-positive specimens, whereas the LC Strep B ASR and BD-StrepB test identified 29.5% and 30.0% positive specimens, respectively. Because of the increased detection rate of 3.0% to 3.5% observed with PCR, a second GBS-specific amplicon was sequenced to confirm the presence of GBS that was not detected by culture. In our hands, the sensitivity/specificity of the LC Strep B ASR was 100%/95.9%, and the BD-StrepB test was 92.5%/92.5% using culture as the gold standard.     

Quantitative detection of Streptococcus pneumoniae from sputum samples with real-time quantitative polymerase chain reaction for etiologic diagnosis of community-acquired pneumonia

We assessed the clinical usefulness of a real-time quantitative polymerase chain reaction (RQ-PCR) method applied on sputum samples to identify Streptococcus pneumoniae in 184 consecutive patients admitted to hospital with community-acquired pneumonia. Induced sputum samples were analyzed by culture and RQ-PCR. In total, 70/184 patients (38%) were diagnosed with S. pneumoniae. Cultures from blood and nasopharyngeal secretions were positive in 27/179 (15%) and 42/158 (27%) cases, respectively. Pneumococcal antigen was detected in 33/169 (20%) urine specimens. In sputum samples, culture was significantly positive in 19/128 (15%), whereas a significant concentration of DNA was found by RQ-PCR in 34/127 (27%) cases (P < 0.001). In 28/34 (82%) patients with RQ-PCR-positive sputum samples, S. pneumoniae was also detected with other methods. In the 34 RQ-PCR-positive sputum samples, 17 were negative by sputum culture, out of which 14 were obtained from patients treated with antibiotics prior to sampling. S. pneumoniae may be rapidly diagnosed by analyzing induced sputum samples by RQ-PCR and may be particularly valuable in patients in whom antibiotic therapy has been initiated.     

Real-time polymerase chain reaction for detection of Isospora belli in stool samples

A real-time polymerase chain reaction (PCR) assay targeting the internal transcribed spacer 2 region of the ribosomal RNA gene was developed for the detection of Isospora belli DNA in fecal samples, including an internal control to detect inhibition during the amplification process. The assay was performed on species-specific DNA controls (n = 27) and a range of positive (n = 21) and negative (n = 120) stool samples, and achieved 100% specificity and sensitivity. The simple fecal sample collection procedure, the high-throughput potential, and the possibility of quantification makes the I. belli real-time PCR assay a powerful diagnostic tool for epidemiologic studies with possibilities for extension to other helminthes and protozoa using additional molecular targets. In addition, this Isospora PCR could augment the clinical laboratory diagnosis of isosporiasis, in particular, in patients with a travel history to developing countries.     

Development of a diagnostic real-time polymerase chain reaction assay for the detection of invasive Haemophilus influenzae in clinical samples

Since the introduction of the Haemophilus influenzae serotype b vaccine, invasive H. influenzae disease has become dominated by nontypeable (NT) strains. Several widely used molecular diagnostic methods have been shown to lack sensitivity or specificity in the detection of some of these strains. Novel real-time assays targeting the fucK, licA, and ompP2 genes were developed and evaluated. The fucK assay detected all strains of H. influenzae tested (n = 116) and had an analytical sensitivity of 10 genome copies/polymerase chain reaction (PCR). This assay detected both serotype b and NT H. influenzae in 12 previously positive specimens (culture and/or bexA PCR) and also detected H. influenzae in a further 5 of 883 culture-negative blood and cerebrospinal fluid (CSF) samples. The fucK assay has excellent potential as a diagnostic test for detection of typeable and nontypeable strains of invasive H. influenzae in clinical samples of blood and CSF.     

提高乙型肝炎病毒P基因区聚合酶链反应检测阳性率的策略

目的：提高乙型肝炎病毒（HBV）DNA P基因区聚合酶链反应（PCR）检测的阳性率。方法：采用优化PCR反应条件，降低退火温度以及采用3'末端碱基游移兼并引物，减少引物与模板引物结合区的错配对PCR的影响等多种方法，对常规PCR检测HBV DNA P基因区阴性的病人血清，再进行PCR检测。结果：通过降低退火温度及减少3'末端错配，PCR检测阳性率由81．7％（67／82）增加至92．7％（76／82，P＜0．05）。结论：综合采用优化PCR反应条件，降低退火温度和采用3'末端碱基游移兼并引物等方法，可提高基因高突变区PCR检测阳性率。     

Carrier detection and feasibility of prenatal diagnosis of hemophilia B by multiplex polymerase chain reaction

Summary Since direct diagnosis based on detection of factor IX mutations by direct sequencing is currently performed in a few laboratories only, restriction fragment length polymorphisms are still useful for carrier detection and prenatal diagnosis of hemophilia B. We analyzed 29 women from 20 families at risk of hemophilia B with three intragenic (MnlI,DdeI,TaqI) and two extragenic (HhaI,BamHI) restriction fragment length polymorphisms, all by the polymerase chain reaction. This analysis confirmed 10 possible carriers and excluded 16 as carriers. The diagnosis was made in 17 of the 20 families (85%). The combination of three polymorphisms indicated thatMnlI/DdeI/HhaI andTaqI/DdeI/HhaI were the most informative (15/20 families, 75% and 14/20 families, 70%). For carrier detection with the most useful polymorphism combination (TaqI/DdeI/HhaI) we devised a multiplex polymerase chain reaction that uses two or three sets of primers, allowing carrier diagnosis in one step with 99% reliability. Our results indicate that the combination of five restriction fragment length polymorphisms makes carrier detection and prenatal diagnosis possible for 85% of families at risk of hemophilia B, with 99% reliability. With multiplex polymerase chain reaction such diagnoses can be obtained faster.     

Use of primers based on the heat shock protein genes hsp70, hsp40, and hsp10, for the detection of bovine mastitis pathogens Streptococcus agalactiae, Streptococcus uberis and Streptococcus bovis

Streptococcus agalactiae, Streptococcus uberis, and Streptococcus bovis are three of the major pathogens which cause mastitis in dairy herds. Since conventional methods for the detection of these mastitis pathogens are laborious and time-consuming, rapid methods are needed. With an attempt to know if heat shock protein (HSP) genes other than HSP60 gene, could be used for PCR primer designing, in this study, we tried to design PCR primers based on the heat shock protein genes hsp70, hsp40, and hsp10 for the specific detection of S. agalactiae, S. uberis, and S. bovis, respectively. Using these primers, all the randomly selected target strains could be specifically detected. Bacterial species other than the target organisms, including strains of other Streptococcus spp., and strains of non-Streptococcus spp., would not generate any false positive results. As these PCR primers were used for direct detection of mastitis pathogens, the detection limit was N (N=1-9) x 10(3)CFU/ml of cell dilutions. If a 10h pre-enrichment step was performed, the detection limit was N x 10(0)CFU/ml. Thus, these primers could be used for the specific and sensitive detection of bovine mastitis bacteria.     

TaqMan荧光定量PCR检测流感嗜血杆菌和肺炎链球菌方法的建立及应用

目的 建立TaqMan荧光定量PCR检测方法 ,用于流感嗜血杆菌和肺炎链球菌的检测和鉴别诊断.方法 针对流感嗜血杆菌种属特异性基因bexA和肺炎链球菌种属特异性基因lytA,设计合成引物和TaqMan探针,研究不同引物和探针荧光定量PCR检测的特异性和灵敏度,确定标本检测中循环阈值(Ct)的临界值(cut-off值).将荧光定量PCR、乳胶凝集和细菌培养3种检测方法 同时应用于278份细菌性脑膜炎患者脑脊髓液标本的检测.结果 bexA基因引物和探针能特异性检测流感嗜血杆菌a、b、c、d血清型的菌株,检测灵敏度为每个反应10个基因组DNA拷贝;lytA基因引物和探针能特异性检测肺炎链球菌常见致病的血清型肺炎链球菌菌株,检测灵敏度为每个反应90个基因组DNA拷贝.通过荧光定量PCR方法 ,278份脑脊髓液标本中共检测出 4份流感嗜血杆菌阳性和7份肺炎链球菌阳性,其中各有2份培养出相应的病原菌,另有1份流感嗜血杆菌和2份肺炎链球菌乳胶凝集结果 阳性.结论 TaqMan荧光定苗PCR方法 能特异地检测和鉴定流感嗜血杆菌和肺炎链球菌,具有较高的灵敏度和快速检测的特点,能提高临床流感嗜血杆菌和肺炎链球菌感染患者标本的阳性检出率.     

A specific polymerase chain reaction test for the identification of Streptococcus pneumoniae

Using an approach based on the comparison of arbitrary primer polymerase chain reaction (PCR) genomic profiles from oral streptococci and Streptococcus pneumoniae strains, we identified a 434-bp genomic fragment apparently specific for S. pneumoniae. From the nucleotidic sequence of this common fragment, a pair of primers was designed and tested on a set of strains comprising the major Streptococcus species. One species, S. anginosus, gave an amplification product of the same length as S. pneumoniae. Sequence comparison of the S. anginosus and S. pneumoniae amplicons revealed several variations which were used to define a new set of primers giving a 181-bp S. pneumoniae-specific fragment. The amplified fragment contains the 5' terminal part of a gene encoding a putative sugar-specific permease and an intergenic sequence. The PCR test was evaluated on 257 strains of invasive S. pneumoniae corresponding to clinical isolates and on 153 non-pneumoniae oral streptococci strains; in addition, 3 S. pseudopneumoniae strains were tested. With these primers, an amplification product was only obtained with the S. pneumoniae strains. Moreover, the test was successfully evaluated on 10 atypical S. pneumoniae strains related to pneumococcal diseases. In this study, we therefore established the capacity of a simple PCR test to discriminate S. pneumoniae from other Streptococci (including S. pseudopneumoniae), thus allowing rapid and accurate diagnosis.     

实时荧光定量PCR检测VAD1 mRNA评估新型隐球菌性脑膜炎的疗效

Objective To establish a new approach for quantitative detection of VAD1 mRNA in cryptococcus neoformans by RT-FQ-PCR, and evaluate the treatment efficacy of CNM. MethodsThe primers and TaqMan probe were designed according to the published sequence of VAD1 mRNA (GenBank),and RT-FQ-PCR method to detect VAD1 mRNA was established. Cerebrospinal fluid from 25 CNM patients and 30 controls were detected and sensitivity and specificity of the method were evaluated. VAD1 mRNA concentration in cerebrospinal fluid from both acute phase and recovery phase of 25 CNM patients were also detected and significance of CNM treatment efficacy with VAD1 mRNA analysis was evaluated. Results Correlation coefficient of standard curve was - 0. 997 9 in detection of VAD1 mRNA by RT-FQ-PCR, and the detection limit was 101 copies/μl. The intra CV of plasmid standard for high, medium and low concentrations were 0. 65% ,0. 89% and 1.23% respectively, the sensitivity of cryptococcus neoformans detection by RT-FQPCR was 96% (24/25) ,while specificity was 100% (30/30). VAD1 mRNA concentration in acute phase were significant higher than that in recovery phase (3. 042 ±0. 906 vs 2. 187 ±0. 665 ,t =4. 583 ,P ＜0. 01).Conclusions The established RT-FQ-PCR method for the detection of VAD1 mRNA is provided with sound sensitivity, specificity and reproducibility, which might be fit for the detection of VAD1 mRNA. The expression level of VAD1 mRNA is relevant with the treatment efficacy of CNM.     

聚合酶链反应检测霍乱弧菌

为快速、准确地检测霍乱弧菌，控制霍乱流行，研究建立了一种聚合酶链反应（ＰＣＲ）检测方法。根据霍乱弧菌肠毒素ＣＴ基因序列，自行设计一对特异引物，扩增片断为２９６ｂｐ，同时采用一种高效率、快速、简便的方法提取并纯化ＤＮＡ，该法能成功地检测各种样品中的产ＣＴ肠毒素Ｏ１群霍乱弧菌。应用ＰＣＲ技术和常规分离培养方法对９０９份腹泻患者粪便标本和１８份环境水样标本进行平行检测。结果表明，两种方法的检测结果一致，粪便标本阳性率为４．２％（３８／９０９）；水样标本为２２．２％（４／１８），其中１份粪便标本和１份水样标本，是经过第２次增菌培养后，检出用性，而ＰＣＲ方法未见假阳性及假阴性结果。表明ＰＣＲ方法准确、快速、灵敏，并可在２、３小时内检测出含３个菌细胞的标本。因此，该方法可广泛应用于检测各种临床、环境标本中的产ＣＴ肠毒素Ｏ１群霍乱弧菌。     

实时荧光定量PCR检测VAD1 mRNA评估新型隐球菌性脑膜炎的疗效

Objective To establish a new approach for quantitative detection of VAD1 mRNA in cryptococcus neoformans by RT-FQ-PCR, and evaluate the treatment efficacy of CNM. MethodsThe primers and TaqMan probe were designed according to the published sequence of VAD1 mRNA (GenBank),and RT-FQ-PCR method to detect VAD1 mRNA was established. Cerebrospinal fluid from 25 CNM patients and 30 controls were detected and sensitivity and specificity of the method were evaluated. VAD1 mRNA concentration in cerebrospinal fluid from both acute phase and recovery phase of 25 CNM patients were also detected and significance of CNM treatment efficacy with VAD1 mRNA analysis was evaluated. Results Correlation coefficient of standard curve was - 0. 997 9 in detection of VAD1 mRNA by RT-FQ-PCR, and the detection limit was 101 copies/μl. The intra CV of plasmid standard for high, medium and low concentrations were 0. 65% ,0. 89% and 1.23% respectively, the sensitivity of cryptococcus neoformans detection by RT-FQPCR was 96% (24/25) ,while specificity was 100% (30/30). VAD1 mRNA concentration in acute phase were significant higher than that in recovery phase (3. 042 ±0. 906 vs 2. 187 ±0. 665 ,t =4. 583 ,P ＜0. 01).Conclusions The established RT-FQ-PCR method for the detection of VAD1 mRNA is provided with sound sensitivity, specificity and reproducibility, which might be fit for the detection of VAD1 mRNA. The expression level of VAD1 mRNA is relevant with the treatment efficacy of CNM.     

Detection of galE gene by polymerase chain reaction in campylobacters associated with Guillain-Barre syndrome

Guillain-Barre Syndrome (GBS) is a neuromuscular disorder and campylobacteriosis is known to trigger the onset of the disorder. A polymerase chain reaction (PCR) protocol was developed that could specifically amplify a 497-bp region of the UDP-galactose 4-epimerase (galE) gene sequence in campylobacters responsible for triggering the onset of GBS. The identity of the PCR product was confirmed by Hind III endonuclease restriction digestion, which produced the predicted 430 and 67-bp DNA fragments. The assay could detect the presence of the gene in Campylobacter suspensions containing as few as 5 cells ml(-1). The assay detected the presence of the gene in 17 of the 20 campylobacters isolated from chicken, 9 of the 13 campylobacters isolated from turkey and 7 of the 7 campylobacters isolated from human stools. All Campylobacter strains isolated from chicken, turkey and clinical samples were resistant to multiple antibiotics. The assay failed to detect the presence of the gene in five different microaerophilic strains of Helicobacter spp., E. coli and Salmonella spp. The entire diagnostic assay, including template preparation, amplification and electrophoresis, can be completed within 6 h.     

A real-time polymerase chain reaction for the detection of Streptococcus pneumoniae in blood using a mouse model: a potential new "gold standard"

Better diagnostics for pneumococcal disease are urgently needed. In a murine model, real-time polymerase chain reaction was superior to conventional culture in detecting pneumococcus in blood, particularly in early disease and after antibiotic administration, and could distinguish between commensalism and infection.     

Identification and characterization of plasmid-borne erm(T) macrolide resistance in group B and group A Streptococcus

One hundred and seven group B Streptococcus (GBS) isolates and 344 group A Streptococcus (GAS) isolates were collected between 2005 and 2009 from 2 area hospitals and studied for resistance to erythromycin (ERY) and clindamycin (CLI) and the presence of the erm(T) macrolide resistance gene. The erm(T) gene was found in 5 (8%) of 61 erythromycin nonsusceptible GBS isolates and in 22 (55%) of 40 erythromycin nonsusceptible GAS isolates. The erm(T) gene in all 27 GBS/GAS erm(T) gene–positive isolates was located on a plasmid. Three erm(T) gene–positive plasmids were DNA sequenced. Two plasmids (1 each from GBS and GAS isolates) were both 4967 bp in size, contained the erm(T) gene, and differed by only 2 base pairs, suggesting interspecies horizontal transfer of the erm(T) gene containing plasmid. The third (GBS) plasmid was 6825 bp in size and contained GBSi1, a group II bacterial intron, as well as the erm(T) gene. Pulsed-field gel electrophoresis of all 27 erm(T) gene containing isolates and a selection of erm(T) gene–negative isolates indicated possible clonal expansion among erm(T) gene containing GAS isolates, but not among the 5 erm(T) gene–positive GBS isolates.     

Cloning and development of synthetic internal amplification control for Bacillus anthracis real-time polymerase chain reaction assays

An internal amplification control (IAC) was developed for Bacillus anthracis rpoB gene detection using TaqMan assay. Synthetic IAC oligonucleotides were subcloned using vector pDG1730 for ectopic integration into host Bacillus subtilis strain 1A772 genome. Differentially labeled target and IAC probes were used in real-time polymerase chain reaction (PCR) assays. There was no nonspecific cross-detection in single-well reactions. Limit of detection for both target and IAC DNA was 5 fg corresponding to a single gene copy. The IAC, in conjunction with target system, should decrease the rate of false-positive and false-negative results in real-time PCR assays.     

肝细胞生长因子mRNA实时荧光定量PCR检测体系的建立及其在淋巴瘤诊断中的价值

目的 构建肝细胞生长因子(HGF)mRNA定量标准,建立实时荧光定量(FQ)-PCR检测体系,探讨其检测淋巴瘤的应用价值.方法 提取总RNA,行逆转录(RT)-PCR,获HGF cDNA目标片段并构建重组质粒,作为定量标准.设计第二引物对和探针,确定扩增条件,优化组分浓度,建立HGF mRNA实时FQ-PCR检测体系,并定量检测47例淋巴瘤患者[霍奇金病(HD)11例,非霍奇金淋巴瘤(NHL)36例;治疗后,获缓解34例,未缓解13例]和19例非肿瘤患者的淋巴组织标本中HGFmRNA表达.同时,用受试者工作特征(ROC)曲线法,评价其在淋巴瘤诊断中的敏感度和特异度.结果 本研究构建的HGF mRNA定量标准和实时FQ-PCR检测体系的标准曲线斜率为-3.513,相关系数为0.999,扩增效率达92.6%;批内、日内和日间变异系数分别为2.1%、4.0%和6.8%;灵敏性达2拷贝/μ1.HGF mRNA在淋巴瘤组的表达(6.425 4-2.172)显著高于非肿瘤对照组(0.317±0.192,t=15.883,P＜0.001),缓解组的表达(6.157±1.712)显著低于未缓解组(7.591±1.184,t=2.768,P＜0.05),但在HD组和NHL组的表达差异无统计学意义(P＜0.05).ROC曲线提示,当临床诊断临界值为3.316时,HGF mRNA对淋巴瘤诊断的敏感度和特异度分别为93.6%和100%.结论 本研究成功构建了HGF mRNA的定量标准和实时FQ-PCR检测体系,并可用于淋巴瘤的HGF mRNA定量检测.     

沙眼衣原体聚合酶链反应测定质控物的构建及其应用研究

目的 研制能够模拟真实临床样本且无生物传染危险性的沙眼衣原体(chlamydia trachomatis,CT)PCR检测质摔物,并进行稳定性研究.方法 在充分查阅文献的基础上,明确国内外已有文献报道的CT:PCR检测的靶序列,选择最普遍应用的CT质粒序列,采用重叠(overlap)PCR、分子克隆等技术构建含有所选择的常用检测目的 CT质粒序列的重组真核表达载体,即pTARGETTM-CT 质粒.然后,将载体以脂质体转染的方式转染入宫颈上皮细胞(HTB-SiHa细胞)中,收集细胞,经含20%小牛血清的细胞培养液稀释,即成为能模拟CT检测临床样本的细胞质控物.最后观察得到的质控物对目前国内常用商品试剂盒的适用性及在4℃、37℃及室温条件下的稳定性.结果 成功构建了含CT质粒(178-610)、(1219-1993)、(2471-3260)、(5239-5864)、(6722-7499)等5个片段的重组真核表达载体,并转入HTB-SiHa细胞中,得到了可模拟临床样本的CT PCR检测的质控物.采用深圳匹基生物工程有限公司、中山大学达安基因股份有限公司商品CT PCR检测试剂盒对转染后样本进行检测,得到阳性结果;定量为3.21×108拷贝/ml;倍比稀释后较高浓度样本检测结果呈梯度下降、10倍稀释循环阈值相差约3.3;对不同浓度稀释后在4℃、37℃及室温条件下保存1个月的样本的检测结果进行随机区组的两因素方差分析后发现,在各温度下保存的样本经检测后的结果问差异无统计学意义,所构建的质控样本至少可以稳定保存1个月.结论 利用重叠PCR与双酶切的方法,建立了一种将多间隔片段连接并构建入同一载体的简便有效的方法.成功地得到了可模拟临床标本的CT PCR检测质控物.     

Differential detection of B. pertussis from B. parapertussis using a polymerase chain reaction (PCR) in presence of SYBR green1 and amplicon melting analysis

Nucleic acid (DNA) from control stock strains of B. pertussis and B. parapertussis (B. pertussis strain # 9797 and B. parapertussis strain # 15234 from ATCC) was amplified by polymerase chain reaction (PCR) targeting pertussis toxin (PT) promotor region, in presence of SYBR green1 a dye that fluoresces on binding specifically to double stranded DNA; and fluorescent melting profile of amplicon (amplified DNA) was studied. Amplicon of B. pertussis and B. parapertussis generated distinctly different melting bands with melting temperature (Tm) at 89.8 and 91.7 degrees C, respectively. Melting profile and Tm of each randomly selected isolates of B. pertussis and B. parapertussis was identical to that of respective control strains. Distinct difference in Tm between B. pertussis and B. parapertussis specific amplicons allowed differential detection of the two Bordetella species based on a single PCR product. The amplified product of serial diluted control stock of bacteria was analyzed by both agarose gel electrophoresis and melting profile analysis. The analytical sensitivity of detection (1-10 CFU equivalent in the tested volume) by melting profile and Tm analysis was in agreement with that obtained by agarose gel analysis.     

Lack of effect of strain type on detection of toxigenic Clostridium difficile by glutamate dehydrogenase and polymerase chain reaction

Glutamate dehydrogenase (GDH) is popular as a preliminary test for the detection of Clostridium difficile. Recent work has suggested that GDH sensitivity may vary according to ribotype and may be lower for ribotypes 002, 027, and 106 compared with polymerase chain reaction (PCR). We investigated this effect using a dilution series of 64 isolates tested by GDH and Cepheid GeneXpert PCR. PCR was significantly more sensitive than GDH overall; however, there was no difference in detection according to specific ribotype.